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Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto‐Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences Presented by Master of Sciences Philipp Konstantin Zimmermann Born in Berlin‐Zehlendorf Oral examination: April 16th, 2015 Genome‐wide detection of induced DNA double strand breaks Referees: Prof. Dr. Christof von Kalle Prof. Dr. Stefan Wiemann I 1. INTRODUCTION 1 1.1 The DNA damage response and genomic instability 1 1.1.1 DNA damaging agents and the DNA damage response 1 1.2 DNA double‐strand break (DSB) repair pathways 3 1.2.1 The Non‐Homologous End Joining (NHEJ) Repair Pathway 4 1.2.2 Methods for the detection of DNA damage and DNA repair activity 6 1.3 Radio‐ and chemotherapy 7 1.3.1 Types of ionizing radiation 7 1.3.2 Radiation‐induced DNA damages 8 1.3.3 The topoisomerase family 9 1.3.4 Topoisomerase 2 catalytic cycle and targeting by anticancer drugs 10 1.3.5 Cancer therapy‐induced delayed genomic instability 11 1.4 Lentiviruses 11 1.4.1 History and phylogeny of lentiviruses 11 1.4.2 The lentiviral genome 12 1.4.3 The life cycle of lentiviruses 13 1.4.4 Structure of lentiviral vectors for gene therapy 15 1.5 Scientific aims 16 2. MATERIALS AND METHODS 18 2.1 Materials 18 2.1.1 Chemicals 18 2.1.2 Enzymes 19 2.1.3 Bacteria 19 2.1.4 Cell lines and primary cells 19 2.1.5 Antibodies 20 2.1.6 Plasmids 20 2.1.7 Oligonucleotides 20 2.1.7.1 Standard primers for q‐RT‐PCR 20 2.1.7.2 Primers used for linker cassettes in LAM‐PCR 21 2.1.7.3 Primers used for LAM‐PCR 21 2.1.7.4 Fusionprimer for Pyrosequencing 21 2.1.7.5 Primers and oligos used for direct DSB labeling approaches 22 2.1.8 Commercial kits 22 2.1.9 Buffers, Media, Solutions 23 2.1.10 Disposables 24 2.1.11 Equipment 24 2.1.12 Software and data bases 25 2.2 Methods 26 II 2.2.1 Cell Culture Methods 26 2.2.1.1 Cell Cultivation 26 2.2.1.2 Freezing and thawing of cells 26 2.2.1.3 Cell Counting 26 2.2.1.4 Transfection 27 2.2.1.5 Transduction 27 2.2.1.6 Virus production 28 2.2.1.7 Determining the lentiviral titer on Hela cells 28 2.2.1.8 MTT Assay 28 2.2.1.9 Immunostaining of H2AX foci 29 2.2.1.10 Inhibition of NHEJ‐repair activity by Nu7441 and Mirin 29 2.2.1.11 Irradiation of cells 30 2.2.1.12 Inhibition of Topoisomerase 2 in mammalian cells with doxorubicin and etoposide 30 2.2.1.13 Preparation for FACS 30 2.2.1.14 Synchronization of NHDF‐A in G1/G0, S and G2/M phase of the cell cycle 31 2.2.2 Molecular Biology Methods 32 2.2.2.1 Isolation of genomic DNA from cultivated cells 32 2.2.2.2 Determining the DNA concentration 32 2.2.2.3 Polymerase Chain Reaction (PCR) 32 2.2.2.4 DNA agarose electrophoresis 33 2.2.2.5 DNA isolation from agarose gel 33 2.2.2.6 Absolute quantitative real‐time PCR (q‐RT‐PCR) 33 2.2.2.7 Linear‐Amplification Mediated Polymerase Chain Reaction (LAM‐PCR) 34 2.2.2.8 Cleaning‐Up of PCR products using AMPure XP beads 39 2.2.2.9 Fusionprimer‐PCR 39 2.2.2.10 SureSelect Target Enrichment for Illumina Multiplexed Sequencing 40 2.2.2.11 Tdt‐mediated labeling of DSB sites 44 2.2.2.12 Biotin Quantification 47 2.2.2.13 Linker‐Amplification‐Mediated DSB Trapping (LAM‐DST) 48 2.2.2.14 Pyrophosphate sequencing 50 2.2.2.15 Cloning of PCR amplicons using the TOPO‐TA Cloning Kit 50 2.2.2.16 Transformation of circular DNA into chemically‐competent E.coli 50 2.2.2.17 Mini‐ and maxipreparation of plasmid DNA 51 2.2.2.18 Enzymatic DNA restriction digest 51 2.2.3 Bioinformatical Methods 51 2.2.3.1 Automated Sequence Analysis (HISAP) 51 2.2.3.2 A549 mRNA expression analysis 52 2.2.3.3 DNaseI Hypersensitive Sites 52 2.2.3.4 Histone modifications and Transcription Factor Binding Sites 52 2.2.3.5 Ingenuity Pathway Analysis (IPA) 53 2.2.3.6 Identification of DSB site clusters in the genome 53 3. RESULTS 54 3.1 IDLV‐mediated capturing of radiation‐induced DSB sites in vivo 54 III 3.1.1 Immunostaining of H2AX foci in irradiated cells 54 3.1.2 MTT assay to determine lethal dose values for etoposide and doxorubicin 55 3.1.3 IDLV‐Delivered DNA‐baits tag radiation‐induced and repaired DSB sites 56 3.1.4 IDLV DSB trapping in NHDF‐A with impaired NHEJ‐repair activity 58 3.1.5 Trapping and mapping of TOP2 poison‐induced DSB 58 3.1.6 Calculating the number of integrated IDLV copies per irradiated cell 59 3.1.7 SureSelect Target Enrichment for analyzing IDLV vector integrity 60 3.2 Analyzing early DSB repair events and kinetics of DSB induction at single nucleotide resolution 61 3.2.1 Genomic tagging of early‐repaired radiation‐induced DSB sites by IDLV 62 3.2.2 Synchronization of NHDF‐A and Hela cells in G1, S and G2 phase 62 3.2.3 Tdt‐mediated labeling of radiation‐induced DSB sites 63 3.2.4 Linker‐Amplification‐Mediated DSB‐Trapping for DSB labeling in real‐time 66 3.3 DSB site distribution in radiation‐surviving and expandable cell populations 68 3.3.1 Identification of radiation‐induced DSB sites by LAM‐PCR 68 3.3.2 DSB are not enriched on chromosomes, in genes and gene‐regulatory regions 69 3.3.3 IDLV integration at radiation‐induced DSB sites is mediated by NHEJ‐repair 71 3.4 Analyzing the influence of transcriptional activity, chromatin status and gene classes and networks on DSB site distribution 72 3.4.1 Trapping of Radiation‐Induced DSB is not influenced by transcriptional activity 72 3.4.2 The location of radiation‐induced DSB sites is composed of histone modifications defining active chromatin 73 3.4.3 Radiation‐induced DSB in radiation‐survivor cells are enriched in genes and networks regulating cell survival 77 3.4.4 Identification and analysis of radiation‐induced DSB sites over time 79 3.5 Identification of frequently damaged and repaired genomic regions 81 3.5.1 DSB Trapping in irradiated and passaged cells reveals common regions of radiation‐induced and repaired damage 81 3.5.2 Radiation‐related DSB hotspots overlap with genes involved in maintaining genome stability and DNA repair 84 3.5.3 DSB hotspots overlap with eu‐ to heterochromatin border regions 86 4. DISCUSSION 88 4.1 Immunostaining of H2AX is not suitable to detect DSB and genomic instabilities in cancer therapy surviving cell populations 88 4.2 NHEJ‐mediated IDLV integration at DSB sites stably marks DNA damage and repair sites in living cells 89 4.3 Identification of radiation‐induced DSB sites 90 4.3.1 Transcriptional activity before irradiation does not influence DSB site distribution 90 4.3.2 DSB site distribution is non‐random with respect to the genome accessibility 91 4.3.3 Genes involved in specific cellular processes are enriched for induced DSB 93 IV 4.3.4 Clonality of DSB site distribution over time 93 4.3.5 Radiation‐induced and repaired DSB sites cluster in hotspots in specific genomic regions 94 4.4 Methods for in situ labeling of induced DSB sites 96 5. SUPPLEMENT 98 5.1 Supplementary Figures 98 5.2 SupplementaryTables 110 5.3 Figure Index 135 5.3.1 Figures 135 5.3.2 Supplementary Figures 136 5.4 Table Index 137 5.4.1 Tables 137 5.4.2 Supplementary Tables 137 5.5 Zusammenfassung 139 5.6 Summary 140 5.7 Abbreviations 141 5.8 References 146 5.9 Publications and congress attendances 152 6. DANKSAGUNG 154 1 INTRODUCTION 1. INTRODUCTION 1.1 The DNA damage response and genomic instability 1.1.1 DNA damaging agents and the DNA damage response The DNA in the cell encodes the genetic information required for the functioning of all cells and living organisms. Every day, the DNA is under constant pressure to be destabilized by various processes and agents. These lesions can block DNA replication and transcription and are broadly divided according to their origin into either endogenous or exogenous (Figure 1) [1]. Endogenous processes such as DNA replication by DNA polymerases induce low levels of DNA damage. Other endogenous sources of DNA damage include genomic fragile sites, nucleases and reactive oxygen species (ROS) such as superoxide and hydrogen peroxide stemming from metabolic processes in the mitochondria [2]. These latter, highly reactive molecules form chemical bonds with other molecules within seconds of their production. Among their target molecules, the DNA is a susceptible target, in which ROS can cause base and sugar modifications, DNA‐protein crosslinks, single‐strand and double‐ strand breaks. In addition to endogenous processes, numerous exogenous sources also threaten the genomic integrity (Figure 1). The most pervasive form of exogenous DNA damaging agent is ultraviolet (UV) rays originating from sunlight that can hit the DNA directly and induce up to 100,000 DNA lesions per exposed cell [2].
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  • Figure S1. 17-Mer Distribution in the Yangtze Finless Porpoise Genome

    Figure S1. 17-Mer Distribution in the Yangtze Finless Porpoise Genome

    Figure S1. 17-mer distribution in the Yangtze finless porpoise genome. The x-axis is 17-mer depth (X); the y-axis is the number of sequencing reads at that depth. Figure S2. Sequence depth distribution of the assembly data. The x-axis shows the sequencing depth (X) and the y-axis shows the number of bases at a given depth. The results demonstrate that 99% of bases sequencing depth is more than 20. Figure S3. Comparison of gene structure characteristics of Yangtze finless porpoise and other cetaceans. The x-axis represents the length of corresponding genetic element of exon number and the y-axis represents gene density. Figure S4. Phylogeny relationships between the Yangtze finless porpoise and other mammals reconstructed by RAxML with the GTR+G+I model. Table S1. Summary of sequenced reads Raw Reads Qualified Reads1 Total Read Sequence Physical Total Read Sequence Physical Library SRA Data Length Coverage2 Coverage2 Data Length Coverage2 Coverage2 Insert Size (bp) Number (Gb) (bp) (×) (×) (Gb) (bp) (×) (×) 289 58.94 150.00 23.67 22.80 57.84 149.75 23.23 22.41 SRR6923836 462 71.33 150.00 28.65 44.12 70.12 149.74 28.16 43.44 SRR6923837 624 67.47 150.00 27.10 56.36 63.90 149.67 25.66 53.50 SRR6923834 791 57.58 150.00 23.12 60.97 55.39 149.67 22.24 58.78 SRR6923835 4,000 108.73 150.00 43.67 582.22 70.74 150.00 28.41 378.80 SRR6923832 7,000 115.4 150.00 46.35 1,081.39 84.76 150.00 34.04 794.27 SRR6923833 11,000 107.37 150.00 43.12 1,581.08 79.78 150.00 32.04 1,174.81 SRR6923830 18,000 127.46 150.00 51.19 3,071.33 97.75 150.00 39.26 2,355.42 SRR6923831 Total 714.28 - 286.87 6,500.27 580.28 - 233.04 4,881.43 - 1Raw reads in mate-paired libraries were filtered to remove duplicates and reads with low quality and/or adapter contamination, raw reads in paired-end libraries were filtered in the same manner then subjected to k-mer-based correction.
  • Preview of “Supplement Table S4.Xls”

    Preview of “Supplement Table S4.Xls”

    Annotations for all recurrently lost regions cytoband 1p36.12 3p14.2 3q13.13 q value 0,01352 0,1513 0,085307 residual q value 0,01352 0,1513 0,085307 wide peak boundaries chr1:23457835-23714048 chr3:60396160-60637030 chr3:110456383-110657226 genes in wide peak E2F2 FHIT DPPA4 ID3 DPPA2 TCEA3 DDEFL1 TBC1D19 PI4K2B STIM2 GBA3 KCNIP4 DKFZp761B107 C4orf28 GPR125 FLJ45721 hsa-mir-218-1 CXCL3 GRSF1 HNRPD HTN1 HTN3 IBSP IGFBP7 IGJ IL8 CXCL10 KDR CXCL9 AFF1 MUC7 NKX6-1 PF4 PF4V1 PKD2 POLR2B PPEF2 PPAT PPBP PRKG2 MAPK10 PTPN13 REST CXCL6 CXCL11 CXCL5 SPINK2 SPP1 SRP72 STATH SULT1E1 UGT2B4 UGT2B7 UGT2B10 UGT2B15 UGT2B17 SPARCL1 VDP SLC4A4 HERC3 GENX-3414 CDKL2 TMPRSS11D ABCG2 ADAMTS3 CLOCK CEP135 G3BP2 HNRPDL ENAM FAM13A1 CXCL13 PAICS UGT2B11 HPSE NMU SMR3B NPFFR2 UGT2A1 CCNI hsa-mir-491 hsa-mir-31 4p15.32 4q13.1 4q13.1 (continued) 6q14.1 0,14888 0,17878 0,093693 0,14888 0,17878 0,093693 chr4:17969802-29966659 chr4:55756035-90966136 chr6:76830186-107898353 CCKAR AFM SEC31A AIM1 DHX15 AFP RUFY3 BCKDHB RBPSUH ALB WDFY3 PRDM1 SOD3 AMBN LPHN3 CCNC SLIT2 ANXA3 DKFZP564O0823 CGA SLC34A2 AREG RCHY1 CNR1 PPARGC1A ART3 ANKRD17 EPHA7 KIAA0746 BMP3 BRDG1 GABRR1 ANAPC4 BTC SMR3A GABRR2 SLA/LP CCNG2 ASAHL GRIK2 LGI2 SCARB2 COQ2 HTR1B TBC1D19 CDS1 SULT1B1 HTR1E PI4K2B CENPC1 TMPRSS11E IMPG1 STIM2 CSN1S1 MRPS18C ME1 GBA3 CSN2 COPS4 NT5E KCNIP4 CSN3 HSD17B11 PGM3 DKFZp761B107 DCK HERC5 POU3F2 C4orf28 DMP1 PLAC8 PREP GPR125 DSPP NUDT9 SIM1 FLJ45721 EPHA5 NUP54 ELOVL4 hsa-mir-218-1 EREG UGT2B28 MAP3K7 FGF5 ODAM TPBG GC HERC6 TTK GK2 SDAD1 RNGTT GNRHR UBE1L2 TBX18